Actin-activated MgATPase Activity Research Articles

A Failure to Communicate: MYOSIN RESIDUES INVOLVED IN HYPERTROPHIC CARDIOMYOPATHY AFFECT INTER-DOMAIN INTERACTION

Our molecular modeling studies suggest a charge-dependent interaction between residues Glu-497 in the relay domain and Arg-712 in the converter domain of human β-cardiac myosin. To test the significance of this putative interaction, we generated transgenic Drosophila expressing indirect flight muscle myosin with charge reversal mutations in the relay (E496R) or converter (R713E). Each mutation yielded dramatic reductions in myosin Ca-ATPase activity (~80%) as well as in basal (~67%) and actin-activated (~84%) Mg-ATPase activity. E496R myosin-induced in vitro actin-sliding velocity was reduced by 71% and R713E myosin permitted no actin motility. Indirect flight muscles of late pupae from each mutant displayed disrupted myofibril assembly, with adults having severely abnormal myofibrils and no flight ability. To understand the molecular basis of these defects, we constructed a putative compensatory mutant that expresses myosin with both E496R and R713E. Intriguingly, ATPase values were restored to ~73% of wild-type and actin-sliding velocity increased to 40%. The double mutation suppresses myofibril assembly defects in pupal indirect flight muscles and dramatically reduces myofibril disruption in young adults. Although sarcomere organization is not sustained in older flies and flight ability is not restored in homozygotes, young heterozygotes fly well. Our results indicate that this charge-dependent interaction between the myosin relay and converter domains is essential to the mechanochemical cycle and sarcomere assembly. Furthermore, the same inter-domain interaction is disrupted when modeling human β-cardiac myosin heavy chain cardiomyopathy mutations E497D or R712L, implying that abolishing this salt bridge is one cause of the human disease.

Regulation of Nonmuscle Myosin II by Tropomyosin Isoforms

Tropomyosin (Tm) regulates actin myosin interactions in eukaryotic cells ranging from yeast to mammalian muscle and nonmuscle cells. Tropomyosin is a α-helical coiled-coil actin binding protein that associates end-to-end to form continuous strands along both sides of the actin filament. Tropomyosins can inhibit or activate actomyosin MgATPase activity and motility depending on the myosin and Tm isoforms. In this study, we have attempted to determine whether activation or inhibition is specified by the Tm or the myosin isoform. We carried out in vitro motility assays with four myosin isoforms (skeletal muscle myosin, and nonmuscle IIA, IIB and IIC HMMs) in the presence of five Tm isoforms (skeletal muscle αTm, and nonmuscle isoforms, Tm2, Tm5a, Tm5NM1 and Tm4) and skeletal muscle actin. With skeletal muscle myosin, actin-Tm filament velocities were inhibited by αTm (∼60%) but activated by Tm5a and Tm5NM1 (30-60%) relative to actin alone, whereas Tm2 and Tm4 had little or no effect. In the case of nonmuscle IIA and IIC HMMs, all nonmuscle Tms activated filament velocities, whereas αTm had no effect, relative to actin alone. None of the Tm isoforms affected filament velocities with nonmuscle IIB HMM. Therefore, the primary determinant of the effect of Tm on actin filament velocities on myosin is the myosin isoform. The actin-activated MgATPase activities of IIA, IIB and IIC HMMs were measured to determine the mechanism of activation by a nonmuscle Tm, Tm5NM1. Tm5NM1 increased the Vmax of both IIA (26%), and IIC (19%), with a much smaller effect on IIB (12%) compared to actin alone thus supporting the motility data. Tm5NM1 also decreased the KATPase of IIA and IIB. Therefore, Tm5NM1 activates the MgATPase activity of IIA and IIB by increasing the Vmax and decreasing KATPase. Supported by NIH.

Characterization of Three Full-length Human Nonmuscle Myosin II Paralogs

Nonmuscle myosin IIs (NM IIs) are a group of molecular motors involved in a wide variety of cellular processes including cytokinesis, migration, and control of cell morphology. There are three paralogs of the NM II heavy chain in humans (IIA, IIB, and IIC), each encoded by a separate gene. These paralogs are expressed at different levels according to cell type and have different roles and intracellular distributions in vivo. Most previous studies on NM II used tissue-purified protein or expressed fragments of the molecule, which presents potential drawbacks for characterizing individual paralogs of the intact protein in vitro. To circumvent current limitations and approach their native properties, we have successfully expressed and purified the three full-length human NM II proteins with their light chains, using the baculovirus/Sf9 system. The enzymatic and structural properties of the three paralogs were characterized. Although each NM II is capable of forming bipolar filaments, those formed by IIC tend to contain fewer constituent molecules than those of IIA and IIB. All paralogs adopt the compact conformation in the presence of ATP. Phosphorylation of the regulatory light chain leads to assembly into filaments, which bind to actin in the presence of ATP. The nature of interactions with actin filaments is shown with different paralogs exhibiting different actin binding behaviors under equivalent conditions. The data show that although NM IIA and IIB form filaments with similar properties, NM IIC forms filaments that are less well suited to roles such as tension maintenance within the cell.

Kinetic Characterization of the ATPase and Actin-activated ATPase Activities of Acanthamoeba castellanii Myosin-2

Phosphorylation of Ser-639 in loop-2 of the catalytic motor domain of the heavy chain of Acanthamoeba castellanii myosin-2 and the phosphomimetic mutation S639D have been shown previously to down-regulate the actin-activated ATPase activity of both the full-length myosin and single-headed subfragment-1 (Liu, X., Lee, D. Y., Cai, S., Yu, S., Shu, S., Levine, R. L., and Korn, E. D. (2013) Proc. Natl. Acad. Sci. U.S.A. 110, E23-E32). In the present study we determined the kinetic constants for each step in the myosin and actomyosin ATPase cycles of recombinant wild-type S1 and S1-S639D. The kinetic parameter predominantly affected by the S639D mutation is the actin-activated release of inorganic phosphate from the acto myosin·ADP·Pi complex, which is the rate-limiting step in the steady-state actomyosin ATPase cycle. As consequence of this change, the duty ratio of this conventional myosin decreases. We speculate on the effect of Ser-639 phosphorylation on the processive behavior of myosin-2 filaments.

The heavy chain has its day

Nonmuscle myosin-II is an actin-based motor that converts chemical energy into force and movement, and thus functions as a key regulator of the eukaryotic cytoskeleton. Although it is established that phosphorylation on the regulatory light chain increases the actin-activated MgATPase activity of the motor and promotes myosin-II filament assembly, studies have begun to characterize alternative mechanisms that regulate filament assembly and disassembly. These investigations have revealed that all three nonmuscle myosin-II isoforms are subject to additional regulatory controls, which impact diverse cellular processes. In this review, we discuss current knowledge on mechanisms that regulate the oligomerization state of nonmuscle myosin-II filaments by targeting the myosin heavy chain.

Regulation of the actin-activated MgATPase activity ofAcanthamoebamyosin II by phosphorylation of serine 639 in motor domain loop 2

It had been proposed previously that only filamentous forms of Acanthamoeba myosin II have actin-activated MgATPase activity and that this activity is inhibited by phosphorylation of up to four serine residues in a repeating sequence in the C-terminal nonhelical tailpiece of the two heavy chains. We have reinvestigated these issues using recombinant WT and mutant myosins. Contrary to the earlier proposal, we show that two nonfilamentous forms of Acanthamoeba myosin II, heavy meromyosin and myosin subfragment 1, have actin-activated MgATPase that is down-regulated by phosphorylation. By mass spectroscopy, we identified five serines in the heavy chains that can be phosphorylated by a partially purified kinase preparation in vitro and also are phosphorylated in endogenous myosin isolated from the amoebae: four serines in the nonhelical tailpiece and Ser639 in loop 2 of the motor domain. S639A mutants of both subfragment 1 and full-length myosin had actin-activated MgATPase that was not inhibited by phosphorylation of the serines in the nonhelical tailpiece or their mutation to glutamic acid or aspartic acid. Conversely, S639D mutants of both subfragment 1 and full-length myosin were inactive, irrespective of the phosphorylation state of the serines in the nonhelical tailpiece. To our knowledge, this is the first example of regulation of the actin-activated MgATPase activity of any myosin by modification of surface loop 2.

Regulation of the filament structure and assembly ofAcanthamoebamyosin II by phosphorylation of serines in the heavy-chain nonhelical tailpiece

Acanthamoeba myosin II (AMII) has two heavy chains ending in a 27-residue nonhelical tailpiece and two pairs of light chains. In a companion article, we show that five, and only five, serine residues can be phosphorylated both in vitro and in vivo: Ser639 in surface loop 2 of the motor domain and serines 1489, 1494, 1499, and 1504 in the nonhelical tailpiece of the heavy chains. In that paper, we show that phosphorylation of Ser639 down-regulates the actin-activated MgATPase activity of AMII and that phosphorylation of the serines in the nonhelical tailpiece has no effect on enzymatic activity. Here we show that bipolar tetrameric, hexameric, and octameric minifilaments of AMII with the nonhelical tailpiece serines either phosphorylated or mutated to glutamate have longer bare zones and more tightly clustered heads than minifilaments of unphosphorylated AMII, irrespective of the phosphorylation state of Ser639. Although antiparallel dimers of phosphorylated and unphosphorylated myosins are indistinguishable, phosphorylation inhibits dimerization and filament assembly. Therefore, the different structures of tetramers, hexamers, and octamers of phosphorylated and unphosphorylated AMII must be caused by differences in the longitudinal stagger of phosphorylated and unphosphorylated bipolar dimers and tetramers. Thus, although the actin-activated MgATPase activity of AMII is regulated by phosphorylation of Ser639 in loop 2 of the motor domain, the structure of AMII minifilaments is regulated by phosphorylation of one or more of four serines in the nonhelical tailpiece of the heavy chain.

Interaction Between the Relay Loop and the SH1-SH2 Helix Region in Drosophila Muscle Myosin is Essential for Normal Motor Function, Myofibril Stability and Muscle Contraction

Molecular modeling of Drosophila muscle myosin reveals that relay domain residue E499 interacts with SH1-SH2 residue R714 in a charge-dependent manner. To explore the significance of this interaction, we generated transgenic lines expressing myosin with a mutation in the relay loop domain (E499R) or the SH1-SH2 helix (R714E). Both mutations yield ∼75% reductions in CaATPase as well as basal or actin-activated MgATPase activity. Actin-sliding velocity of E499R is reduced by 65% and R714E shows no motility. Indirect flight muscles in late pupae of each mutant display disrupted myofibril assembly. Two-hour- and two-day-old adults have severely abnormal myofibril morphology and poor sarcomere organization, with no flight ability. Therefore, the putative interaction of the relay with SH1-SH2 is indispensable for normal motor function, myofibril stability and locomotion. We next constructed a putative compensatory mutant designed to restore function of the E499R or R714E mutant by generating a transgenic line that expresses both E499R and R714E. Interestingly, calcium, basal, and actin-stimulated ATPase values are restored to 65–70% of wild type and actin-sliding velocity is at 40%. The compensatory mutation suppresses the assembly defects in pupal muscle that are seen in both E499R and R714E and reduces disrupted myofibril morphology in two-hour old indirect flight muscles. However, the E499R-R714E mutant myosin was unable to maintain sarcomere organization in two-day-old flies or to restore flight ability. Overall, our results reveal that interaction of residues at the relay/SH1-SH2 helix interface is important for myosin and muscle function.

Identification of Calmodulin and MlcC as Light Chains for Dictyostelium Myosin-I Isozymes

Dictyostelium discoideum express seven single-headed myosin-I isozymes (MyoA-MyoE and MyoK) that drive motile processes at the cell membrane. The light chains for MyoA and MyoE were identified by expressing Flag-tagged constructs consisting of the motor domain and the two IQ motifs in the neck region in Dictyostelium. The MyoA and MyoE constructs both copurified with calmodulin. Isothermal titration calorimetry (ITC) showed that apo-calmodulin bound to peptides corresponding to the MyoA and MyoE IQ motifs with micromolar affinity. In the presence of calcium, calmodulin cross-linked two IQ motif peptides, with one domain binding with nanomolar affinity and the other with micromolar affinity. The IQ motifs were required for the actin-activated MgATPase activity of MyoA but not MyoE; however, neither myosin exhibited calcium-dependent activity. A Flag-tagged construct consisting of the MyoC motor domain and the three IQ motifs in the adjacent neck region bound a novel 8.6 kDa two EF-hand protein named MlcC, for myosin light chain for MyoC. MlcC is most similar to the C-terminal domain of calmodulin but does not bind calcium. ITC studies showed that MlcC binds IQ1 and IQ2 but not IQ3 of MyoC. IQ3 contains a proline residue that may render it nonfunctional. Each long-tailed Dictyostelium myosin-I has now been shown to have a unique light chain (MyoB-MlcB, MyoC-MlcC, and MyoD-MlcD), whereas the short-tailed myosins-I, MyoA and MyoE, have the multifunctional calmodulin as a light chain. The diversity in light chain composition is likely to contribute to the distinct cellular functions of each myosin-I isozyme.

Drosophila melanogaster Myosin-18 Represents a Highly Divergent Motor with Actin Tethering Properties

The gene encoding Drosophila myosin-18 is complex and can potentially yield six alternatively spliced mRNAs. One of the major features of this myosin is an N-terminal PDZ domain that is included in some of the predicted alternatively spliced products. To explore the biochemical properties of this protein, we engineered two minimal motor domain (MMD)-like constructs, one that contains the N-terminal PDZ (myosin-18 M-PDZ) domain and one that does not (myosin-18 M-ΔPDZ). These two constructs were expressed in the baculovirus/Sf9 system. The results suggest that Drosophila myosin-18 is highly divergent from most other myosins in the superfamily. Neither of the MMD constructs had an actin-activated MgATPase activity, nor did they even bind ATP. Both myosin-18 M-PDZ and M-ΔPDZ proteins bound to actin with Kd values of 2.61 and 1.04 μm, respectively, but only about 50–75% of the protein bound to actin even at high actin concentrations. Unbound proteins from these actin binding assays reiterated the 60% saturation maximum, suggesting an equilibrium between actin-binding and non-actin-binding conformations of Drosophila myosin-18 in vitro. Neither the binding affinity nor the substoichiometric binding was significantly affected by ATP. Optical trapping of single molecules in three-bead assays showed short lived interactions of the myosin-18 motors with actin filaments. Combined, these data suggest that this highly divergent motor may function as an actin tethering protein.

The Enzymatic Motor Activity of Nonmuscle Myosin II-B is not Critical for Cardiac Myocyte Cytokinesis

Nonmuscle myosin (NM) II is believed to drive contractile ring constriction during cytokinesis and NM II-B is the major nonmuscle myosin II isoform expressed in cardiac myocytes. Ablation of NM II-B in mice resulted in premature binucleation in cardiac myocytes consistent with a role for NM II-B in cytokinesis. Surprisingly binucleation can be rescued by expressing a motor-impaired mutant form of NM II-B (R709C) in the mouse heart, suggesting that NM II-B enzymatic motor activity is not essential for cytokinesis. We tested this hypothesis directly by using a COS-7 cell line which resembles cardiac myocytes in containing only NM II-B and small quantities of NM II-C (<7%) but no NM II-A. Previous work had shown that using siRNA to deplete NMHC II-B in this cell line resulted in multinucleation due to a failure in cytokinesis. We now show that COS-7 cell multinucleation can be rescued by transfecting with mutant forms of NM II-B (R709C) or NM II-A (N93K). These two mutant NM IIs have previously been shown to have marked reductions in their actin-activated MgATPase activity (70% in the case of NM II-B and 96% in the case of NM II-A) and neither could propel actin filaments in an in vitro motility assay. Importantly, cytokinesis is inhibited by the myosin II inhibitor blebbistatin in COS-7 cells expressing either wild-type NM II-B or the mutant form of NM II-A (or II-B). Whereas blebbistatin blocks myosin in an actin-detached state, the mutant NM IIs (R709C II-B and N93K II-A) have no defect in their actin-binding activity. Our results therefore argue that the role of NM II in cytokinesis depends more on its actin binding (cross-linking) property than its enzymatic Mg2+-ATPase activity.

Nonmuscle myosin IIA with a GFP fused to the N-terminus of the regulatory light chain is regulated normally

Nonmuscle myosin II plays a crucial role in a variety of cellular processes (e.g., polarity formation, cell motility, and cytokinesis). It is composed of two heavy chains, two regulatory light chains and two essential light chains. The ATPase activity of the myosin II motor domain is regulated through phosphorylation of the regulatory light chain (RLC) by myosin light chain kinase. To study myosin function and localization in cellular processes, GFP-fused RLCs are widely used; however, the exact kinetic properties of myosins with bound GFP-RLC are poorly described. More importantly, it has not been shown that a regulatory light chain fused at its N-terminus with GFP can maintain the normal phosphorylation-dependent regulation of nonmuscle myosin or serve as a substrate for myosin light chain kinase. We coexpressed N-terminal GFP-RLC with a heavy meromyosin (HMM)-like fragment of nonmuscle myosin IIA and essential light chain to characterize the phosphorylation dynamics and in vitro kinetic properties of the resulting HMM. Myosin light chain kinase phosphorylates the GFP-RLC bound to HMM IIA with the same V(max) as it does the wild type RLC bound to HMM IIA, but the K(m) is about two fold higher for the GFP fusion protein, meaning that it is a somewhat poorer substrate. The steady-state actin-activated MgATPase activity of the GFP-RLC HMM is very low in the absence of phosphorylation demonstrating that the GFP moiety does not prevent formation of the off state. The actin-activated MgATPase activity of phosphorylated GFP-RLC-HMM and is about half that of wild type phosphorylated HMM. The ability of phosphorylated GFP-RLC-HMM to move actin filaments in the actin gliding assay is also slightly compromised. These data indicate that despite some kinetic differences the N-terminal GFP fusion to the regulatory light chain is a reasonable model system for studying myosin function in vivo.

HGAL, a Lymphoma Prognostic Biomarker, Regulates Lymphocyte and Lymphoma Cell Motility by Modulation of RhoA Signaling Pathway.

Abstract 316Identification of genes whose expression correlates with clinical outcome is of paramount importance for designing upfront patient-tailored treatments that will potentially improve the outcome of lymphoma patients. Furthermore, new molecular prognostic biomarkers may lead to the discovery of novel pathophysiological mechanisms underlying lymphoma pathogenesis, potentially highlighting unique biological processes regulated by these proteins in lymphoma cells and in their normal cellular counterparts. HGAL is a novel germinal center (GC)-specific gene whose expression predicts survival of patients with diffuse large B-cell lymphoma (DLBCL) and classical Hodgkin lymphoma (cHL). Recently, we have demonstrated that HGAL is involved in negative regulation of lymphocyte migration, thus potentially constraining lymphocytes to the GC and preventing lymphoma dissemination; however, the molecular mechanism of HGAL effects on cell motility is unknown. Herein, we demonstrated that HGAL serves as a physiological regulator of the RhoA signaling pathway. HGAL over-expression or siRNA-mediated knock-down leads to increased or decreased levels of GTP-bound RhoA, respectively, but does not affect levels of GTP-bound CDC42 and RAC1. HGAL-induced activation of RhoA results in inhibition of lymphocyte and lymphoma cell motility and chemotaxis by activation of its downstream effector ROCK leading to: a) phosphorylation of myosin regulatory light chain (MRLC) that regulates actin-activated Mg-ATPase activity of myosin II; b) phosphorylation of myosin phosphatase (myosin PPTase) subunit MYPT1 inhibiting myosin light chain dephosphorylation; and c) phosphorylation of cofilin leading to cytoskeletal reorganization, as reflected by increased formation of focal adhesions and stress fibers. Opposite effects on RhoA downstream effectors are observed upon knock-down of HGAL expression. Furthermore, HGAL over-expression or knock-down modulate additional functions of RhoA, including transcriptional activation by serum response factor and RhoA transforming potential, as assessed by foci formation in NIH 3T3 cells. Co-immunoprecipitation experiments demonstrated that the effect of HGAL on RhoA is indirect and is mediated by RhoA-specific guanine nucleotide exchange factors (RhoGEFs) PDZ-RhoGEF and LARG that stimulate the GDP-GTP exchange rate. Moreover, we delineate the structural domains of HGAL and PDZ-RhoGEF that mediate the interaction between these proteins. Over-expression of HGAL mutants lacking the PDZ binding domain, mediating the interaction between HGAL and these RhoGEFs, fails to induce activation of RhoA and does not inhibit lymphoma cell motility and chemotaxis. Similarly, knock-downs of PDZ-RhoGEF and/or LARG reverse the inhibitory effects of HGAL on lymphoma cell motility and chemotaxis. These observations reveal a novel molecular mechanism underlying the inhibitory effects of HGAL on the motility of normal GC lymphocyte and GC-derived lymphoma cells. Since HGAL is specifically expressed only during the GC stage of B lymphocyte differentiation, these findings highlight the uniqueness and complexity of cell processes occurring during the GC reaction. They further elucidate the pathophysiologic mechanism underlying the favorable outcome of DLBCL and cHL patients whose tumors express high levels of HGAL protein. Disclosures:No relevant conflicts of interest to declare.

An Alternatively Spliced Isoform of Non-muscle Myosin II-C Is Not Regulated by Myosin Light Chain Phosphorylation

We report a novel isoform of non-muscle myosin II-C (NM II-C), NM II-C2, that is generated by alternative splicing of an exon, C2, encoding 41 amino acids in mice (33 in humans). The 41 amino acids are inserted into loop 2 of the NM II-C heavy chain within the actin binding region. Unlike most vertebrate non-muscle and smooth muscle myosin IIs, baculovirus-expressed mouse heavy meromyosin (HMM) II-C2 demonstrates no requirement for regulatory myosin light chain (MLC(20)) phosphorylation for maximum actin-activated MgATPase activity or maximum in vitro motility as measured by the sliding actin filament assay. In contrast, noninserted HMM II-C0 and another alternatively spliced isoform HMM II-C1, which contains 8 amino acids inserted into loop 1, are dependent on MLC(20) phosphorylation for both actin-activated MgATPase activity and in vitro motility ( Kim, K. Y., Kovacs, M., Kawamoto, S., Sellers, J. R., and Adelstein, R. S. (2005) J. Biol. Chem. 280, 22769-22775 ). HMM II-C1C2, which contains both the C1 and C2 inserts, does not require MLC(20) phosphorylation for full activity similar to HMM II-C2. These constitutively active C2-inserted isoforms of NM II-C are expressed only in neuronal tissue. This is in contrast to NM II-C1 and NM II-C0, both of which are ubiquitously expressed. Full-length NM II-C2-GFP expressed in COS-7 cells localizes to filaments in interphase cells and to the cytokinetic ring in dividing cells.

Direct association of calponin with specific domains of PKC-α

Calponin contributes to the regulation of smooth muscle contraction through its interaction with F-actin and inhibition of the actin-activated Mg-ATPase activity of phosphorylated myosin. Previous studies have shown that the contractile agonist acetylcholine induced a direct association of translocated calponin and PKC-alpha in the membrane. In the present study, we have determined the domain of PKC-alpha involved in direct association with calponin. In vitro binding assay was carried out by incubating glutathione S-transferase-calponin aa 92-229 with His-tagged proteins of individual domains and different combinations of domains of PKC-alpha. Calponin was found to bind directly to the full-length PKC-alpha. Calponin bound to C2 and C4 domains but not to C1 and C3 domains of PKC-alpha. When incubated with proteins of different combination of domains, calponin bound to C2-C3, C3-C4, and C2-C3-C4 but not to C1-C2 or C1-C2-C3. To determine whether these in vitro bindings mimic the in vivo associations, and in vivo binding assay was performed by transfecting colonic smooth muscle cells with His-tagged proteins of individual domains and different combinations of domains of PKC-alpha. Coimmunoprecipitation of calponin with His-tagged truncated forms of PKC-alpha showed that C1-C2, C1-C2-C3, C2-C3, and C3-C4 did not associate with calponin. Calponin associated only with full-length PKC-alpha and with C2-C3-C4 in cells in the resting state, and this association increased upon stimulation with acetylcholine. These data suggest that calponin bound to fragments that may mimic the active form of PKC-alpha and that the functional association of PKC-alpha with calponin requires both C2 and C4 domains during contraction of colonic smooth muscle cells.

Functional Effects of the Hypertrophic Cardiomyopathy R403Q Mutation Are Different in an α- or β-Myosin Heavy Chain Backbone

The R403Q mutation in the beta-myosin heavy chain (MHC) was the first mutation to be linked to familial hypertrophic cardiomyopathy (FHC), a primary disease of heart muscle. The initial studies with R403Q myosin, isolated from biopsies of patients, showed a large decrease in myosin motor function, leading to the hypothesis that hypertrophy was a compensatory response. The introduction of the mouse model for FHC (the mouse expresses predominantly alpha-MHC as opposed to the beta-isoform in larger mammals) created a new paradigm for FHC based on finding enhanced motor function for R403Q alpha-MHC. To help resolve these conflicting mechanisms, we used a transgenic mouse model in which the endogenous alpha-MHC was largely replaced with transgenically encoded beta-MHC. A His(6) tag was cloned at the N terminus of the alpha-and beta-MHC to facilitate protein isolation by Ni(2+)-chelating chromatography. Characterization of the R403Q alpha-MHC by the in vitro motility assay showed a 30-40% increase in actin filament velocity compared with wild type, consistent with published studies. In contrast, the R403Q mutation in a beta-MHC backbone showed no enhancement in velocity. Cleavage of the His-tagged myosin by chymotrypsin made it possible to isolate homogeneous myosin subfragment 1 (S1), uncontaminated by endogenous myosin. We find that the actin-activated MgATPase activity for R403Q alpha-S1 is approximately 30% higher than for wild type, whereas the enzymatic activity for R403Q beta-S1 is reduced by approximately 10%. Thus, the functional consequences of the mutation are fundamentally changed depending upon the context of the cardiac MHC isoform.

The B2 alternatively spliced isoform of nonmuscle myosin II-B lacks actin-activated MgATPase activity and in vitro motility

We report the initial biochemical characterization of an alternatively spliced isoform of nonmuscle heavy meromyosin (HMM) II-B2 and compare it with HMM II-B0, the nonspliced isoform. HMM II-B2 is the HMM derivative of an alternatively spliced isoform of endogenous nonmuscle myosin (NM) II-B, which has 21-amino acids inserted into loop 2, near the actin-binding region. NM II-B2 is expressed in the Purkinje cells of the cerebellum as well as in other neuronal cells [X. Ma, S. Kawamoto, J. Uribe, R.S. Adelstein, Function of the neuron-specific alternatively spliced isoforms of nonmuscle myosin II-B during mouse brain development, Mol. Biol. Cell 15 (2006) 2138–2149]. In contrast to any of the previously described isoforms of NM II (II-A, II-B0, II-B1, II-C0 and II-C1) or to smooth muscle myosin, the actin-activated MgATPase activity of HMM II-B2 is not significantly increased from a low, basal level by phosphorylation of the 20kDa myosin light chain (MLC-20). Moreover, although HMM II-B2 can bind to actin in the absence of ATP and is released in its presence, it cannot propel actin in the sliding actin filament assay following MLC-20 phosphorylation. Unlike HMM II-B2, the actin-activated MgATPase activity of a chimeric HMM with the 21-amino acid II-B2 sequence inserted into the homologous location in the heavy chain of HMM II-C is increased following MLC-20 phosphorylation. This indicates that the effect of the II-B2 insert is myosin heavy chain specific.

Calcium and cargoes as regulators of myosin 5a activity

Myosin 5a is a two-headed actin-dependent motor that transports various cargoes in cells. Its enzymology and mechanochemistry have been extensively studied in vitro. It is a processive motor that takes multiple 36nm steps on actin. The enzymatic activity of myosin 5 is regulated by an intramolecular folding mechanism whereby its lever arms fold back against the coiled-coil tail such that the motor domains directly bind the globular tail domains. We show that the structure seen in individual folded molecules is consistent with electron density map of two-dimensional crystals of the molecule. In this compact state, the actin-activated MgATPase activity of the molecule is markedly inhibited and the molecule cannot move processively on surface bound actin filaments. The actin-activated MgATPase activity of myosin 5a is activated by increasing the calcium concentration or by binding of a cargo-receptor molecule, melanophilin, in vitro. However, calcium binding to the calmodulin light chains results in dissociation of some of the calmodulin which disrupts the ability of myosin 5a to move on actin filaments in vitro. Thus we propose that the physiologically relevant activation pathway in vivo involves binding of cargo-receptor proteins.

Characterization of Native Myosin VI Isolated from Sea Urchin Eggs

Myosin VI is a molecular motor that is ubiquitously expressed among eukaryotic cells, and thought to be involved in membrane trafficking and anchoring the organelle to actin cytoskeleton. Studies on myosin VI have been carried out using recombinant proteins, but native myosin VI has not been purified yet. Here we purified native myosin VI from sea urchin eggs and characterized its properties. We found that the native myosin VI was a monomeric and non-processive motor protein, and also showed that it moved toward the pointed end of F-actin. Ca2+ stimulated actin-activated MgATPase activity of the native myosin VI, while it lowered its motility on F-actin. Immunofluorescence microscopy showed that the myosin VI was translocated from the inner cytoplasm to the cortex after fertilization. Myosin VI may be involved in endocytic activities in fertilized eggs.

Three-dimensional structure of the myosin V inhibited state by cryoelectron tomography

Unconventional myosin V (myoV) is an actin-based molecular motor that has a key function in organelle and mRNA transport, as well as in membrane trafficking. MyoV was the first member of the myosin superfamily shown to be processive, meaning that a single motor protein can 'walk' hand-over-hand along an actin filament for many steps before detaching. Full-length myoV has a low actin-activated MgATPase activity at low [Ca2+], whereas expressed constructs lacking the cargo-binding domain have a high activity regardless of [Ca2+] (refs 5-7). Hydrodynamic data and electron micrographs indicate that the active state is extended, whereas the inactive state is compact. Here we show the first three-dimensional structure of the myoV inactive state. Each myoV molecule consists of two heads that contain an amino-terminal motor domain followed by a lever arm that binds six calmodulins. The heads are followed by a coiled-coil dimerization domain (S2) and a carboxy-terminal globular cargo-binding domain. In the inactive structure, bending of myoV at the head-S2 junction places the cargo-binding domain near the motor domain's ATP-binding pocket, indicating that ATPase inhibition might occur through decreased rates of nucleotide exchange. The actin-binding interfaces are unobstructed, and the lever arm is oriented in a position typical of strong actin-binding states. This structure indicates that motor recycling after cargo delivery might occur through transport on actively treadmilling actin filaments rather than by diffusion.